C. elegans ATG-5 mutants associated with ataxia

Intercellular cleaning via autophagy is crucial for maintaining cellular homeostasis, and impaired autophagy has been associated with the accumulation of protein aggregates that can contribute to neurological diseases. Specifically, the loss-of-function mutation in the human autophagy-related gene 5 (ATG5) at E122D has been linked to the pathogenesis of spinocerebellar ataxia in humans. In this study, we generated two homozygous C. elegans strains with mutations (E121D and E121A) at positions corresponding to the human ATG5 ataxia mutation to investigate the effects of ATG5 mutations on autophagy and motility. Our results showed that both mutants exhibited a reduction in autophagy activity and impaired motility, suggesting that the conserved mechanism of autophagy-mediated regulation of motility extends from C. elegans to humans.

(f) The frequency of body bends on adult days 3 and 7 in WT, atg-5(E121D) and atg-5(E121A) (N=30) animals. Plots show the frequencies of body bends. Horizontal lines and error bars indicate means ± s.d.

Description
Autophagy is an intracellular degradation system that operates constitutively. This system selectively degrades abnormal intracellular molecules, such as aggregated proteins and impaired organelles, in response to their production (Yamamoto et al., 2023). Animals deficient in autophagy-related genes (ATGs), which are required to drive autophagy, are affected by neurodegenerative diseases with accumulation of abnormal proteins (Hara et al., 2006). These indicate that intracellular cleaning via autophagy contributes to the suppression of disease development through the maintenance of homeostasis.
The autophagy pathway is based on large-scale membrane trafficking, particularly the generation and elongation of isolation membranes that sequester substrates. ATG5 is one of the most well-known ATGs involved in sequestration membrane elongation. Specifically, ATG5 catalyzes the ATG8 lipidation reaction through covalent binding with ATG12 for the recruitment of ATG8 on the isolation membrane (Mizushima et al., 2011). Note that ATG5 is functionally and structurally conserved from humans to C. elegans (Fig. 1a). A pathogenic ATG5 E122D mutation leading to human spinocerebellar ataxia reduces both the interaction with ATG12 and autophagy induction (Kim et al., 2016). However, there are insufficient studies on the pathogenesis of spinocerebellar ataxia due to a defect in the autophagy machinery. To address this issue, we generated a homozygous nematode strain with an atg-5 mutation at a position corresponding to a human ataxia mutation (atg-5(tj130[E121D])). We also generated an atg-5 mutant in which Glu121 was converted to alanine (atg-5(tj122[E121A])) to investigate the importance of carboxylate side chain of the glutamate residue. The side chain of alanine, the methyl group, is sterically small and does not engage in hydrogen bonding or ionic interactions. In both atg-5 mutants, no marked differences in body length, brood size, and growth rate from wild-type worms were observed (Fig. 1b−d).
To observe autophagy levels in these mutants, we utilized GFP-tagged LGG-1 (GFP::LGG-1), a C. elegans ATG8 ortholog, that is widely accepted as an autophagosome marker (Mizushima, 2004). In worms in which autophagy is induced, GFP::LGG-1 is detected as dot-like structures in cells (Kang et al., 2007). In both atg-5 mutants, the number of GFP::LGG-1 dots in the pharynx was lower than wild-type worms, suggesting a permanent decrease in autophagy (Fig. 1e).
A marked decrease in locomotion ability has been observed in patients with spinocerebellar ataxia (Kim et al., 2016). We tested whether a similar phenotype was observed in atg-5(tj130[E121D]) or atg-5(tj122[E121A]), using body bend frequency to evaluate the locomotion ability of worms (Onken & Driscoll, 2010). At day 7 of adulthood, both atg-5(E121D) and atg-5(E121A) worms showed a statistically significant reduction in body bend frequency (Fig. 1f). As early as day 3 of adulthood, a tendency of reduced frequency of body bend was observed in the atg-5(E121A) mutants (Fig. 1f). It is interesting because, to our knowledge, such alanine mutant has not been reported in human ataxia. Collectively, these results indicate that the E121 mutation on ATG-5 reduced the locomotion ability of worms that might be related to the human spinocerebellar ataxia.
In summary, we introduced an ATG5 mutation associated with human spinocerebellar ataxia into C. elegans for the first time and demonstrated that this mutation causes locomotor defects in the nematode. Our analysis of GFP::LGG-1 dots indicated that the two mutant strains created in this study had defective autophagy activity, but their impact on locomotion appeared to be somewhat different. This nematode model could be used to investigate the etiology and pathogenesis of spinocerebellar degeneration in humans.

Body length
L4 larvae were collected, and 16-18 hours later, animals were taken under a microscope and measured as 1-day adults. At least 28 worms were analyzed.

Brood size
Individual L4 stage worms (N=9) were transferred every 12 hours. The number of fertilized eggs and hatched larvae were counted repeatedly until the eggs were laid unfertilized.

Growth rate
After performing timed-egg-lay of 10 adult worms at 20°C, collecting approximately 100 eggs, the number of worms at each stage (egg, L1, L2−L3, L4, adult) was counted every 12 hours.

Measurement of frequency of body bends
The assay was performed as previously described (Onken & Driscoll, 2010). Thirty worms in 20 µL of M9 buffer were transferred to a glass slide. After 2 minutes, the behavior of the worms in the M9 buffer was recorded for 30 seconds. Body bends were counted by reviewing the 30−second movies (N=30).

Statistical analysis
Statistical analyses were performed using Dunnett's test. In all tests, P-value of < 0.05 was considered statistically significant.